Keratinocyte Viability Research Articles

Anthraquinone Derivatives and Other Aromatic Compounds from Marine Fungus Asteromyces cruciatus KMM 4696 and Their Effects against Staphylococcus aureus.

New anthraquinone derivatives acruciquinones A-C (1-3), together with ten known metabolites, were isolated from the obligate marine fungus Asteromyces cruciatus KMM 4696. Acruciquinone C is the first member of anthraquinone derivatives with a 6/6/5 backbone. The structures of isolated compounds were established based on NMR and MS data. The absolute stereoconfigurations of new acruciquinones A-C were determined using ECD and quantum chemical calculations (TDDFT approach). A plausible biosynthetic pathway of the novel acruciquinone C was proposed. Compounds 1-4 and 6-13 showed a significant antimicrobial effects against Staphylococcus aureus growth, and acruciquinone A (1), dendryol B (4), coniothyrinone B (7), and ω-hydroxypachybasin (9) reduced the activity of a key staphylococcal enzyme, sortase A. Moreover, the compounds, excluding 4, inhibited urease activity. We studied the effects of anthraquinones 1, 4, 7, and 9 and coniothyrinone D (6) in an in vitro model of skin infection when HaCaT keratinocytes were cocultivated with S. aureus. Anthraquinones significantly reduce the negative impact of S. aureus on the viability, migration, and proliferation of infected HaCaT keratinocytes, and acruciquinone A (1) revealed the most pronounced effect.

IL-22–Induced Ubiquitin-Specific Protease 15 Promotes Proliferation and Inflammation of Keratinocytes through Stabilization of Squamous Cell Carcinoma Antigen 2

Ubiquitin-specific protease 15 (USP15) plays a significant role in regulating various biological processes in several autoimmune diseases and cancers. However, its role in psoriatic keratinocytes (KCs) has not been extensively studied. In this study, we described that USP15 promotes proliferation and inflammation in KCs by stabilizing squamous cell carcinoma antigen 2. We discovered that the expression of USP15 and squamous cell carcinoma antigen 2 was elevated in lesions from patients with clinical psoriasis and an imiquimod-induced psoriatic dermatitis mouse model. USP15 was able to bind, deubiquitinate, and stabilize squamous cell carcinoma antigen 2. Knocking down USP15 resulted in reduced KC inflammation and impaired KC viability and clonogenicity. Topically applying USP15 small interfering RNA significantly ameliorated imiquimod-induced psoriatic dermatitis and reduced the infiltration of T cells and neutrophils. In addition, we determined that IL-22 was a key cytokine that upregulated the expression of USP15. These findings provide insights regarding the mechanisms involved in the proliferation and inflammation of KCs mediated by IL-22, suggesting a potential IL-22-USP15-squamous cell carcinoma antigen 2 axis in the pathogenesis of psoriatic KCs.

STUDY OF CYTOCOMPATIBILITY OF DIFFERENT SUPPLEMENTS IN GEL COMPOSITIONS USED FOR THE TREATMENT OF PERIODONTAL DISEASES IN ORTHODONTIC PATIENTS VIA EXPLORATION OF MECHANISMS OF THEIR CYTOPATHIC AND CYTOPROTECTIVE EFFECTS IN CULTURED HUMAN AND MOUSE CELLS

Introduction. A search continues for effective means which may reduce the overload of harmful factors, eliminate the inflammatory process, and reduce stress on the periodontal tissues during the active period of orthodontic treatment. We developed and patented the gel composition (GC) Benzidaflaziverdine prepared based on Proteflazid® (flavonoids) and benzydamine hydrochloride (BH) T-Sept® for the local treatment of the periodontal tissues in the form of a periodontal dressing in the orthodontic patients.
 The aim of this study was to evaluate the cytocompatibility of different combinations of components in gel composition based on flavonoid complex and benzydamine hydrochloride (Benzidaflaziverdine) used for the treatment of periodontal diseases in orthodontic patients. For this, mechanisms of their cytopathic and cytoprotective effects will be explored using cultured human and mouse cells.
 Methods. We studied the effect of different supplements used in GC Benzidaflaziverdine on the viability of pseudonormal human keratinocytes of the HaCaT line and mouse fibroblasts of the BALB-3T3 line, and mouse macrophages of the J774.2 line. Various methods of cell survival assessment were used: MTT-assay, staining of cells with fluorescent dyes Hoechst 33342 and Propidium iodide (PI), as well as a test for the genotoxic effects on cells (DNA comet assay). The antioxidant properties of the developed GC variants were evaluated using DPPH (1,1-diphenyl-2-picrylhydrazyl), Merck (Dam-stadt, Germany), and DCFDA-H2 (2’,7’-dichlorodihydrofluorescein diacetate).
 Results. We demonstrated that the Sample containing gel base and BH in the form of a solution (Tantum Verde®) possessed weak prooxidant properties. While the Sample contained gel base, powdered BH (T-Sept®) and Sample containing gel base and powdered BH (T-Sept® and Proteflazid®) possessed pronounced antioxidant properties.
 Conclusions. Tests with DPPH and DCFDA dyes were used to confirm the hypothesis regarding the cytoprotective effect of the patented gel composition Benzidaflaziverdine for local application in the form of a periodontal bandage due to the antioxidant activity of the flavonoid complex, which reaches the maximum level at the 2nd hour of exposure. This gel composition can be recommended for use in clinical periodontology for medical support of orthodontic patients before and during the active phase of orthodontic treatment.

Antibacterial effect and cell metabolic activity of Na2CaSi2O6, β-NaCaPO4, and β-NaCaPO4-SiO2versus hydroxyapatite

Hydroxyapatite (Ca5(PO2)3(OH)) is extensively used in diverse clinical applications because of its relatively simple synthesis protocol, low cost, and chemical similarity to the mineral component of bones and teeth. Some limitations regarding bioactivity and antibacterial activity have motivated changes in its structure and the development of alternative materials to achieve better performances as bioceramics. In this study, the antibacterial effect and cell metabolic activity of the main crystalline phases (Na2CaSi2O6, β-NaCaPO4, and β-NaCaPO4−SiO2) derived from the original 45S5 bioactive glass were evaluated and compared with those induced by a commercial powder of hydroxyapatite. Powder samples of Na2CaSi2O6, β-NaCaPO4, and β-NaCaPO4−SiO2 were synthesized by a simple and reproducible solid-state reaction method. The β-NaCaPO4−SiO2 sample exhibited the strongest effect against Pseudomonas aeruginosa, Staphylococcus aureus, and S. epidermidis, which are bacteria that normally colonize the skin but can become opportunistic pathogens when the host's resistance is low. All bacteria were eliminated within 5 min in the direct-contact test, an effect that is likely associated with changes in pH in the microenvironment generated from the partial solubilization of the material and the action of certain released ions, such as Ca2+. In vitro experiments with human cells, no sample showed cytotoxicity, and the β-NaCaPO4−SiO2 sample stood out once more inducing the most positive effect on keratinocyte and stem cell viability. Overall, the tested materials demonstrated similar or better (β-NaCaPO4−SiO2) biochemical properties than hydroxyapatite. This finding encourages us, and perhaps other research groups, to assess the bio-performance of these materials more comprehensively, aiming at the development of new products for use in tissue engineering.

Photoprotection Against UV-Induced Skin Damage Using Hyaluronic Acid Produced by Lactiplantibacillus plantarum and Enterococcus durans

Exposure to ultraviolet (UV) radiation is one of the major factors that causes skin aging, erythema, sunburns, and skin cancer. This study aimed to select probiotic bacterial isolates able to produce high yield of hyaluronic acid (HA) to be employed for skin photoprotection and other possible biological applications. The selected isolates K11 and St3 were able to produce the highest yields of HA 4.8 and 4.4 mg/ml, respectively. Both isolates were identified as Enterococcus durans strain K11 and Lactiplantibacillus plantarum strain St3 using 16S rRNA gene sequencing. The antioxidant activity of HA produced by E. durans strain K11 and L. plantarum strain St3 was (65.4 0.2%) and (66.6 0.1%), respectively. The viability of UVB-irradiated keratinocytes pre-treated with HA produced by E. durans strain K11 and L. plantarum strain St3 was 91.3 and 91.4%, respectively, compared with the control. While the viability of UVB-irradiated keratinocytes post-treated with HA produced by E. durans strain K11 and L. plantarum strain St3 was 86 and 88.5%, respectively. To the best of our knowledge, this is the first recordation of HA production by Enterococcus durans and Lactiplantibacillus plantarum which revealed a significant radioprotection of the human keratinocytes against UVB radiation.

Evaluation of Toxicological Risks of Nail Coatings Containing Acrylate Monomer HEMA

Individuals working in nail salons are exposed to 2-hydroxyethyl methacrylate (HEMA). HEMA has been found to have several effects on the skin: skin itching, peeling, redness and allergic contact dermatitis. The purpose of the study was to compare the effects of nail coatings containing HEMA and 2-hydroxypropyl methacrylate (HPMA) on the skin. In this study we explored the irritation properties of HEMA and HPMA containing nail coatings in cell cultures in-vitro and in skin PATCH tests under dermatological control. The cytotoxicity of coatings was tested in BALB/c3T3 and HaCaT cell lines by a neutral red uptake assay. Cytotoxicity was expressed as a concentration-dependent reduction of the uptake of neutral red, compared to the untreated controls. Open patch tests were supervised by a certified dermatologist. Polymerized coating extracts have little effect on Balb/c 3T3 cell viability, while having mild cytotoxic effects on HaCaT keratinocytes. Among two tested samples, extracts of HEMA containing coating exhibited higher cytotoxicity – reduction of keratinocyte viability by 28.29 % in case of undiluted 24 h extract and even by 48.26 % in case of 50 % extract was observed. Coating cytotoxiaty observed on HaCaT showed that the keratinocyte cell line was more sensitive to HEMA than to HPMA containing coating.

Study on the Key Genes and Molecular Mechanisms of IL-27 Promoting Keratinocytes Proliferation Based on Transcriptome Sequencing.

IL-27 involves psoriasis pathogenesis potentially by promoting excessive keratinocyte proliferation. However, the underlying mechanisms remain unclear. This study aims to explore the key genes and molecular mechanisms of IL-27-induced keratinocyte proliferation. Primary keratinocytes and immortalized human keratinocyte HaCaT cells were treated with different concentrations of IL-27 for 24 h and 48 h respectively. CCK-8 assay was used to detect cell viability and Western blot was used to detect the expression of CyclinE and CyclinB1. Primary keratinocytes and HaCaT cells were treated with IL-27, and their differentially expressed (DE) genes were obtained by transcriptome sequencing. Then Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed to predict related pathways, and the long non-coding RNA-microRNA-messenger RNA network and protein-protein interaction network were constructed to screen key genes. Biochemical experiments were performed to assess the content of glucose (Glu), lactic acid (LA), and ATP. Flow cytometry and Mito-Tracker Green staining were used to detect mitochondrial membrane potential and the number of mitochondria respectively. Western blot was performed to assess the expression of glucose transporter 1 (GLUT1), hexokinase 2 (HK2), lactate dehydrogenase A (LDHA), phosphoglycerate kinase 1 (PGK1), phosphorylated dynamin-related protein 1 (p-DRP1) (s637) and mitofusin 2 (MFN2). IL-27 concentration-dependently increased keratinocyte viability and the expression of CyclinE and CyclinB1. Bioinformatics analysis showed that the enriched pathways of DE genes were closely associated with cellular metabolism. miR-7-5p, EGFR, PRKCB, PLCB1 and CALM3 were key genes. IL-27 increased the content of LA, mitochondrial membrane potential, and the expression of GLUT1, HK2, LDHA, PGK1, p-DRP1 (s637), and MFN2, accompanied by decreased contents of Glu and ATP (P<0.001). IL-27 potentially promotes keratinocyte proliferation by enhancing glycolysis, mitochondrial function, and mitochondrial fusion. The findings of this study may be conducive to revealing the role of IL-27 in the pathogenesis of psoriasis.

Development of Salmon Sperm DNA/Regenerated Silk Bio-Based Films for Biomedical Studies on Human Keratinocyte HaCaT Cells under Solar Spectrum.

In this study, we fabricated adhesive patches from silkworm-regenerated silk and DNA to safeguard human skin from the sun's rays. The patches are realized by exploiting the dissolution of silk fibers (e.g., silk fibroin (SF)) and salmon sperm DNA in formic acid and CaCl2 solutions. Infrared spectroscopy is used to investigate the conformational transition of SF when combined with DNA; the results indicated that the addition of DNA provides an increase in the SF crystallinity. UV-Visible absorption and circular dichroism spectroscopy showed strong absorption in the UV region and the presence of B-form of DNA once dispersed in the SF matrix, respectively. Water absorption measurements as well as thermal dependence of water sorption and thermal analysis, suggested the stability of the fabricated patches. Biological results on cellular viability (MTT assay) of keratinocyte HaCaT cells after exposures to the solar spectrum showed that both SF and SF/DNA patches are photo-protective by increasing the cellular viability of keratinocytes after UV component exposure. Overall, these SF/DNA patches promise applications in wound dressing for practical biomedical purposes.

Inflammatory stimulation of osteoblasts and keratinocytes from a SAPHO patient for implant risk evaluation

The present report exemplifies a translational method, which could assist the clinical pre-evaluation of patients at risk before surgical interventions. In this study, a presurgical implant decision in a case of SAPHO (synovitis, acne, palmoplantar pustulosis, hyperostosis, osteitis) is described. Since the etiology of this syndrome is likely to involve genetic, infectious and immunological components, lipopolysaccharides (LPS) may conceptually trigger cytokine production leading to a specific chronic inflammation and immunological host response. This may hamper proper healing or accentuate the destruction of periodontal host tissues. In our approach, we examined the ex vivo cell viability and immune responses of primary osteoblasts and keratinocytes under sterile inflammation induced by P. gingivalis LPS. Keratinocytes and osteoblasts were obtained from biopsies of the keratinized gingiva and alveolar bone tissues of a SAPHO human subject. Enzymatically dissociated cells were thus cultured and incubated to LPS at different concentrations (50ng/ml, 200ng/ml, 500ng/ml and 1µg/ml) for 24 h in order to test inflammatory cytokine response (quantitative real time PCR) and toxicity (cell viability). Healthy primary keratinocytes and osteoblasts were used as control cells. The highest concentration of LPS (1µg/ml) significantly reduced cell viability (p &lt; 0.05). Meanwhile, all tested LPS concentrations similarly enhanced the mRNA expressions of selected inflammatory cytokines (TNFalpha, IL-6, IL-8, IL-1beta and IL-1alpha) up to =~3.5-fold, when compared to the healthy cell controls (p &lt; 0.05). This study demonstrated a valuable inflammatory risk evaluation before implant placement, which was successfully performed based on the presented laboratory diagnostic/prognostic approach.&#x0D; Accepted for publication: December 12, 2022&#x0D; Published online: January, 2022

TFAP2C exacerbates psoriasis-like inflammation by promoting Th17 and Th1 cells activation through regulating TEAD4 transcription.

Psoriasis is one of the chronic and autoimmune skin diseases. It is important to uncover the mechanisms underlying the psoriasis. Transcription factor activator protein (TFAP-2) gamma, also known as AP2-gamma, is a protein encoded by the TFAP2C gene. Immune-mediated pathophysiological processes could be linked to psoriasis, but the mechanism is still unclear. Therefore, to date the cause of psoriasis has not been understood completely. Psoriasis is a complex disease triggered by genetic, immunological, and environmental stimuli. Keratinocytes play an important role in both initiation and maintenance phases of psoriasis. A psoriatic keratinocyte model was established by stimulating high sensitivity of human epidermal keratinocytes (HaCaT) to topoisomerase inhibitor cell lines using the accumulation of M5 cytokines comprising interleukin (IL)-17A, IL-22, oncostatin M, IL-1α, and tumor necrosis factor-α (TNF-α). The TFAP2C and transcriptional enhanced associate domain 4 (TEAD4) genes expression was evaluated by reverse transcription-quantitative polymerase chain reaction. Western blot analysis was used to examine protein expression. Cell viability (quantitative) of keratinocytes, including cytotoxicity, proliferation, and cell activation, was evaluated by the MTT assay. The relative percentage values of interleukin (IL)-17a, interferon gamma, and IL-4+ cells were measured by flow cytometry. Accordingly, chromatin immunoprecipitation and luciferase reporter assays were applied to evaluate the binding affinity of TFAP2C and TEAD4 promoter. Level of the TFAP2C gene was elevated in the lesional skin of psoriasis patients. On the other hand, silencing of the TFAP2C gene suppressed the proliferation and inflammatory response in M5-induced keratinocytes. In addition, inhibition of TFAP2C alleviated imiquimod (IMQ)-induced skin injury in mice model. We also observed that suppression of TFAP2C inhibited the activation of T-helper 17 (Th17) and Th1 cells in IMQ-induced mice model. Mechanically, TFAP2C promoted TEAD4 transcriptional activation. TFAP2C exacerbated psoriasis-like inflammation by increasing the activation of Th17 and Th1 cells by regulating TEAD4 transcription. This finding clearly indicated that TFAP2C could be considered a valuable biomarker for the prevention and treatment for psoriasis.

Marine Collagen-Based Bioink for 3D Bioprinting of a Bilayered Skin Model.

Marine organisms (i.e., fish, jellyfish, sponges or seaweeds) represent an abundant and eco-friendly source of collagen. Marine collagen, compared to mammalian collagen, can be easily extracted, is water-soluble, avoids transmissible diseases and owns anti-microbial activities. Recent studies have reported marine collagen as a suitable biomaterial for skin tissue regeneration. The aim of this work was to investigate, for the first time, marine collagen from basa fish skin for the development of a bioink for extrusion 3D bioprinting of a bilayered skin model. The bioinks were obtained by mixing semi-crosslinked alginate with 10 and 20 mg/mL of collagen. The bioinks were characterised by evaluating the printability in terms of homogeneity, spreading ratio, shape fidelity and rheological properties. Morphology, degradation rate, swelling properties and antibacterial activity were also evaluated. The alginate-based bioink containing 20 mg/mL of marine collagen was selected for 3D bioprinting of skin-like constructs with human fibroblasts and keratinocytes. The bioprinted constructs showed a homogeneous distribution of viable and proliferating cells at days 1, 7 and 14 of culture evaluated by qualitative (live/dead) and qualitative (XTT) assays, and histological (H&E) and gene expression analysis. In conclusion, marine collagen can be successfully used to formulate a bioink for 3D bioprinting. In particular, the obtained bioink can be printed in 3D structures and is able to support fibroblasts and keratinocytes viability and proliferation.

Pickering emulsions stabilized with biodegradable nanoparticles for the co-encapsulation of two active pharmaceutical ingredients

Innovative Pickering emulsions co-encapsulating two active pharmaceutical ingredients (API) were formulated for a topical use. An immunosuppressive agent, either cyclosporine A (CysA) or tacrolimus (TAC), was encapsulated at high drug loading in biodegradable and biocompatible poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP). These NP stabilized the oil droplets (Miglyol) containing an anti-inflammatory drug, calcitriol (CAL). The influence of the API on the physico-chemical properties of these emulsions were studied. Emulsions formulated with or without API had a similar macroscopic and microscopic structure, as well as interfacial properties, and they exhibited a good stability for at least 55 days. The emulsions did not alter the viability of human keratinocytes (HaCaT cell line) after 2 and 5 days of exposure to NP concentrations equivalent to efficient API dosages. Thus, these new Pickering emulsions appear as a promising multidrug delivery system for the treatment of chronical inflammatory skin diseases.

Secretome of Adipose-Derived Stem Cells Cultured in Platelet Lysate Improves Migration and Viability of Keratinocytes.

Chronic wounds depict a silent epidemic challenging medical professionals worldwide. Regenerative medicine uses adipose-derived stem cells (ADSC) in promising new therapies. In this study, platelet lysate (PL) as a xenogen-free substitute for foetal bovine serum (FBS) in ADSC culture was used to create an ADSC secretome containing cytokines for optimal wound healing conditions. The ADSC secretome was tested on keratinocytes for migrational behaviour and viability. Therefore, human ADSC were characterized under FBS (10%) and PL (5% and 10%) substitution, regarding morphology, differentiation, viability, gene and protein expression. ADSC were then cultured in 5% PL and their secretome was used for stimulation of keratinocyte migration and viability. To enhance the effect, ADSC were treated with Epithelial Growth Factor (EGF, 100 ng/mL) and hypoxia (1% O₂). In both PL and FBS groups, ADSC expressed typical stem cell markers. PL induced a significantly higher increase in cell viability compared to FBS substitution. ADSC secretome contained various beneficial proteins which enhance the wound healing capacity of keratinocytes. This could be optimized treating ADSC with hypoxia and EGF. In conclusion, the study shows that ADSC cultivated in 5% PL can effectively support wound healing conditions and can be considered as a promising new therapy for individual treatment of chronic wound disorders.

Potential Use of Brazilian Green Propolis Extracts as New Photosensitizers for Antimicrobial Photodynamic Therapy against Cariogenic Microorganisms

The synergic effect of Streptococcus mutans and Candida albicans increases dental caries severity. Antimicrobial photodynamic therapy (aPDT) is a non-invasive treatment for antimicrobial aspects. However, the current photosensitizers (PS) have many downsides for dental applications. This study aimed to evaluate the efficiency of two different Brazilian green propolis (BGP-AF and BGP-AG) as PS for aPDT against these microorganisms. A single-species biofilm was irradiated with crude extracts and their fractions and controls. Such extracts showed the best results and were evaluated in dual-species biofilms. Photodegradation, reactive oxygen species (ROS), cytotoxicity, and color stability assays were also investigated. Reductions higher than 3 log10 CFU/mL (p < 0.0001) occurred for crude BGP in single- and dual-species biofilms. Singlet oxygen was produced in BGP (p < 0.0001). BGP-mediated aPDT delayed S. mutans and C. albicans regrowth after 24 h of treatment (p < 0.0001). Both BGP did not change the color of dental materials (p > 0.05). BGP-AF-mediated aPDT showed 72.41% of oral keratinocyte viability (p < 0.0001). BGP extracts may be used in aPDT against S. mutans and C. albicans. Specifically, BGP-AF may represent a promising PS for dental applications.

Potential of the Ethyl Acetate Fraction of Padina boergesenii as a Natural UV Filter in Sunscreen Cream Formulation

Brown seaweeds, due to their wide range of bioactive compounds, have a high ability to inhibit free radicals and protect against ultraviolet rays. In the present study, the ethyl acetate fraction (EF) was isolated from the Padina boergesenii brown seaweed. Antioxidant activity (by the DPPH scavenging activity method) and cytotoxicity against UVB-induced cytotoxicity in HaCaT human keratinocytes were evaluated. Then, this fraction was used as a bio-filter in the formulation of sunscreen, and the physical properties and stability were investigated. The results showed that the EF could inhibit DPPH radical scavenging (54 ± 1%) and cell viability of HaCaT keratinocytes exposed to UVB irradiation (81.2 ± 0.1%). The results of the stability study of the cream formulated with EF showed that at temperatures 4 °C and 25 °C it has high stability; and at 40 °C on the 28th day, a slight decrease in its stability was observed. The pH and Sun Protection Factor of the cream formulated with EF were reported at 5.8 and 20.55, respectively. Also, the DPPH scavenging activity of the cream was not altered for 28 days of storage at temperatures of 4-40 °C. According to our results, it was proved that the sunscreen formulated with EF of P. boergesenii brown seaweed has promising properties and characteristics that can create a new opportunity for the development of cosmetics and skin care products.

Effect of Zinc Ions on the Proliferation and Differentiation of Keratinocytes

Background: Psoriasis is a chronic inflammatory disease in which there is hyperproliferation and abnormal differentiation of keratinocytes. Since high levels of KLK7, an enzyme inhibited by zinc (Zn2+) ions, are present in psoriatic lesions, we have studied the effect of zinc ions in the viability of keratinocytes, as well as in the activity of KLK5 and KLK7 and in the expression of epidermal markers. Methods and Results: The cells were cultured in the absence or presence of Zn2+ ions (5.0, 10 and 25 M). Cell viability was evaluated by the MTT method after during 14 days. Cell death was evaluated by flow cytometry using propidium iodide. The activity of the KLK was evaluated on the hydrolysis of synthetic substrates. Expression of involucrin, filaggrin, cytokeratins (CK) 5, 10 and 14 was evaluated by quantitative PCR. Cell incubation with Zn2+ ions did not result in significant changes in cell viability. By MTT assay, it was observed that the cultures incubated with 10 and 25 M Zn2+ ions showed a decrease in the number of viable cells in comparison to the control. Cells cultured for 1 day in the presence of 25 M Zn2+ ions displayed a decrease in KLK7 activity. In the presence of Zn2+ ions, it was shown an increase in the expression of CK5, 10 and 14, involucrin and filaggrin. Conclusions: These results have shown that zinc ions can affect the differentiation of HaCat cells, contributing for future therapeutic trials related to psoriasis based on the modulation of KLK activity.

Role of geneticin in isolation and culturing of skin melanocytes and melanoma cells

Abstract Introduction Development of an effective, suitable, and reliable method for both the isolation and culturing of melanocytes is crucial for studies on pathomechanisms of skin diseases originating from melanocyte disorders. In this study, we have investigated the influence of geneticin (G418), a substance used for melanocyte selection, in the view of the frequency of presence of cells such as keratinocytes and fibroblasts, widely known as contaminators of melanocyte-originating cell cultures. Materials and Methods Study was conducted on primary, freshly isolated melanocytes, keratinocytes, fibroblasts, and melanoma cells as well as on commercially available melanoma cell lines MeWo, G-361, and A375. Cells were cultured in different culture media supplemented with various concentrations of geneticin ranging from 0.05 to 1 mg/mL. Cell viability, proliferation rate and detection of apoptotic/necrotic cells was assessed. Results Choice of culture media supplemented with various concentrations of geneticin (0.05 mg/mL, 0.1 mg/mL, 0.5 mg/mL and 1 mg/mL) strongly affect viability of melanocytes, fibroblasts, and keratinocytes. Selective culture media without FBS facilitate the process of melanocytes and melanoma pure cell culture, yet without geneticin supplementation are insufficient for complete eradication of fibroblast contamination from cell culture. Conclusions In this study we provide, for the first time, the dose-response action of keratinocytes and fibroblasts upon geneticin stimulation in different culture media and show that a low concentration (0.05 mg/mL) of geneticin added to the selective culture media may be safely implemented to facilitate the production of melanocyte and melanoma cell cultures that are free from frequent cell contaminants.

Pitavastatin Induces Apoptosis of Cutaneous Squamous Cell Carcinoma Cells through Geranylgeranyl Pyrophosphate-Dependent c-Jun N-Terminal Kinase Activation.

Pitavastatin is a cholesterol-lowering drug and is widely used clinically. In addition to this effect, pitavastatin has shown the potential to induce apoptosis in cutaneous squamous cell carcinoma (SCC) cells. The purpose of this study is to investigate the effects and possible action mechanisms of pitavastatin. SCC cells (SCC12 and SCC13 cells) were treated with pitavastatin, and induction of apoptosis was confirmed by Western blot. To examine whether pitavastatin-induced apoptosis is related to a decrease in the amount of intermediate mediators in the cholesterol synthesis pathway, the changes in pitavastatin-induced apoptosis after supplementation with mevalonate, squalene, geranylgeranyl pyrophosphate (GGPP) and dolichol were investigated. Pitavastatin dose-dependently induced apoptosis of cutaneous SCC cells, but the viability of normal keratinocytes was not affected by pitavastatin at the same concentrations. In supplementation experiments, pitavastatin-induced apoptosis was inhibited by the addition of mevalonate or downstream metabolite GGPP. As a result of examining the effect on intracellular signaling, pitavastatin decreased Yes1 associated transcriptional regulator and Ras homolog family member A and increased Rac family small GTPase 1 and c-Jun N-terminal kinase (JNK) activity. All these effects of pitavastatin on signaling molecules were restored when supplemented with either mevalonate or GGPP. Furthermore, pitavastatin-induced apoptosis of cutaneous SCC cells was inhibited by a JNK inhibitor. These results suggest that pitavastatin induces apoptosis of cutaneous SCC cells through GGPP-dependent JNK activation.

Anti-cariogenic property of Carica papaya, Trachyspermum ammi, Caesalpinia crista linn extracts and their effect on human oral keratinocytes.

Dental caries is a dynamic and composite process. The multifactorial etio-pathogenesis thus influences the initiation and the progression of the disease. The prime pathogenic bacterium includes Streptococcus mutans, Lactobacillus sp and Actinomyces viscosus. The purpose of this in vitro study was to analyze the antimicrobial property of the test herbal extracts and also their effects on the human oral keratinocytes. The bacterial strains S. mutans (American Type Culture Collection [ATCC]-25175); Lactobacillus species (ATCC 4356) and A. viscosus (ATCC 15987) were cultured in the specific culture media-Mitis Salivarius Bacitracin, Man Rogosa Sharpe and Enrichment media, respectively. The test extracts were exposed to the cultured plates and the mean zone of inhibition was measured. The test herbal extracts were also tested for deleterious effects on oral keratinocytes via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Independent Student's t-test and analysis of variances were performed.-25175); Lactobacillus species (ATCC 4356) and A. viscosus (ATCC 15987) were cultured in the specific culture media-Mitis Salivarius Bacitracin, Man Rogosa Sharpe and Enrichment media, respectively. The test extracts were exposed to the cultured plates and the mean zone of inhibition was measured. The test herbal extracts were also tested for deleterious effects on oral keratinocytes via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Independent Student's t-test and analysis of variances were performed. The extracts of Carica papaya, Trachyspermum ammi and Caesalpinia crista linn inhibited the growth of bacteria and the antimicrobial effect was found to be statistically significant at the neat/standard concentration (100 μg/ml). The three extracts showed a cell viability range 96%-99% indicating that the test extracts did not produce or display any deleterious effects on the oral keratinocytes. The three test herbal extracts possess effective anti-cariogenic properties with near par with the efficacy of chlorhexidine and T. ammi proved to be the most potent. The extracts at different concentrations also proved to be safe, noncytotoxic producing a range of 96%-99% of cell viability of the oral keratinocytes.

Photoprotective Activity of Plants Used for Skin Disorders by the Native Population from the Brazilian Amazon Basin: A Preliminary Study

Pharmacognosy Research,2023,15,1,112-118.DOI:10.5530/097484900267Published:December 2022Type:Original ArticleAuthors:Thiago Portal, Margarete Tavares, Marcieni Ataide, Nadia Rezende Barbosa, Joseane R da Silva, Waldiney Pires Moraes, and Jose Luiz Vieira Author(s) affiliations:Thiago Portal, Margarete Tavares, Marcieni Ataide, Nadia Rezende Barbosa, Joseane R da Silva, Waldiney Pires Moraes, Jose Luiz Vieira* Faculty of Pharmacy, Campus Universitario do Guama, Para Federal University, Belem-PA, Brazil. Abstract:Background: Sunscreens are essential in tropical countries with a high annual incidence of solar radiation. The incorporation of natural products in their formulations has great public acceptance and prevents several issues related to organic and inorganic filters, such as adverse reactions. The present study evaluates the antioxidant and photoprotective activities and the ability to inhibit tyrosinase of three plant species traditionally used by Amazonian inhabitants in the treatment of skin disorders. Materials and Methods: The antioxidant activity, the capacity to inhibit the enzyme tyrosinase, ratio UVA/UVB, critical wavelength, sun protection factor, and cellular viability was assessed in ethanolic extracts at 10% of Chrysobalanus icaco, Kalanchoe pinnata, and Ayapana triplinervis collected in the State of Para-Brazil. Results: Chrysobalanus icaco and Kalanchoe pinnata showed promising antioxidant activity. Ayapana triplinervis demonstrated considerable inhibition of the tyrosinase enzyme. Plants extracts had low protection against UVB radiation, but adequate UVA protection. Ayapana triplinervis presented the lowest cellular viability of keratinocytes and murine fibroblasts. Conclusion: The addition of these extracts to other natural products with high sun protection factors can provide the consumer with a natural sunscreen. Keywords:Antioxidant, Ayapana triplinervis, Chrysobalanus icaco, Depigmenting agent, Kalanchoe pinnata, Photoprotection, Skin disordersView:PDF (246.16 KB)